Realistic measurement uncertainties for marine macronutrient measurements conducted using gas segmented flow and Lab-on-Chip techniques.

Accurate and precise measurements of marine macronutrient concentrations are fundamental to our understanding of biogeochemical cycles in the ocean. Quantifying the measurement uncertainty associated with macronutrient measurements remains a challenge. Large systematic biases (up to 10%) have been identified between datasets, restricting the ability of marine biogeochemists to distinguish between the effects of environmental processes and analytical uncertainty. In this study we combine the routine analyses of certified reference materials (CRMs) with the application of a simple statistical technique to quantify the combined (random + systematic) measurement uncertainty associated with marine macronutrient measurements using gas segmented flow techniques. We demonstrate that it is realistic to achieve combined uncertainties of ~1-4% for nitrate + nitrite (ΣNOx), phosphate (PO43-) and silicic acid (Si(OH)4) measurements. This approach requires only the routine analyses of CRMs (i.e. it does not require inter-comparison exercises). As CRMs for marine macronutrients are now commercially available, it is advocated that this simple approach can improve the comparability of marine macronutrient datasets and therefore should be adopted as 'best practice'. Novel autonomous Lab-on-Chip (LoC) technology is currently maturing to a point where it will soon become part of the marine chemist's standard analytical toolkit used to determine marine macronutrient concentrations. Therefore, it is critical that a complete understanding of the measurement uncertainty of data produced by LoC analysers is achieved. In this study we analysed CRMs using 7 different LoC ΣNOx analysers to estimate a combined measurement uncertainty of < 5%. This demonstrates that with high quality manufacturing and laboratory practices, LoC analysers routinely produce high quality measurements of marine macronutrient concentrations.

Buzz off! An evaluation of ultrasonic acoustic vibration for the disruption of marine micro-organisms on sensor-housing materials.

Biofouling is a process of ecological succession which begins with the attachment and colonization of micro-organisms to a submerged surface. For marine sensors and their housings, biofouling can be one of the principle limitations to long-term deployment and reliability. Conventional antibiofouling strategies using biocides can be hazardous to the environment, and therefore alternative chemical-free methods are preferred. In this study, custom-made testing assemblies were used to evaluate ultrasonic vibration as an antibiofouling process for marine sensor-housing materials over a 28-day time course. Microbial biofouling was measured based on (i) surface coverage, using fluorescence microscopy and (ii) bacterial 16S rDNA gene copies, using Quantitative polymerase chain reaction (PCR). Ultrasonic vibrations (20 KHz, 200 ms pulses at 2-s intervals; total power 16·08 W) significantly reduced the surface coverage on two plastics, poly(methyl methacrylate) and polyvinyl chloride (PVC) for up to 28 days. Bacterial gene copy number was similarly reduced, but the results were only statistically significant for PVC, which displayed the greatest overall resistance to biofouling, regardless of whether ultrasonic vibration was applied. Copper sheet, which has intrinsic biocidal properties was resistant to biofouling during the early stages of the experiment, but inhibited measurements made by PCR and generated inconsistent results later on. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, ultrasonic acoustic vibration is presented as a chemical-free, ecologically friendly alternative to conventional methods for the perturbation of microbial attachment to submerged surfaces. The results indicate the potential of an ultrasonic antibiofouling method for the disruption of microbial biofilms on marine sensor housings, which is typically a principle limiting factor in their long-term operation in the oceans. With increasing deployment of scientific apparatus in aquatic environments, including further offshore and for longer duration, the identification and evaluation of novel antifouling strategies that do not employ hazardous chemicals are widely sought.

A multi-parametric assessment of decontamination protocols for the subglacial Lake Ellsworth probe.

Direct measurement and sampling of pristine environments, such as subglacial lakes, without introducing contaminating microorganisms and biomolecules from the surface, represents a significant engineering and microbiological challenge. In this study, we compare methods for decontamination of titanium grade 5 surfaces, the material extensively used to construct a custom-made probe for reaching, measuring and sampling subglacial Lake Ellsworth in West Antarctica. Coupons of titanium were artificially contaminated with Pseudomonas fluorescens bacteria and then exposed to a number of decontamination procedures. The most effective sterilants were (i) hydrogen peroxide vapour, and (ii) Biocleanse™, a commercially available, detergent-based biocidal solution. After each decontamination procedure the bacteria were incapable of proliferation, and showed no evidence of metabolic activity based on the generation of adenosine triphosphate (ATP). The use of ultraviolet irradiation or ethyl alcohol solution was comparatively ineffective for sterilisation. Hydrogen peroxide vapour and ultraviolet irradiation, which directly damage nucleic acids, were the most effective methods for removing detectable DNA, which was measured using 16S rRNA gene copy number and fluorescence-based total DNA quantification. Our results have not only been used to tailor the Ellsworth probe decontamination process, but also hold value for subsequent engineering projects, where high standards of decontamination are required.

Microbiology: lessons from a first attempt at Lake Ellsworth.

During the attempt to directly access, measure and sample Subglacial Lake Ellsworth in 2012-2013, we conducted microbiological analyses of the drilling equipment, scientific instrumentation, field camp and natural surroundings. From these studies, a number of lessons can be learned about the cleanliness of deep Antarctic subglacial lake access leading to, in particular, knowledge of the limitations of some of the most basic relevant microbiological principles. Here, we focus on five of the core challenges faced and describe how cleanliness and sterilization were implemented in the field. In the light of our field experiences, we consider how effective these actions were, and what can be learnt for future subglacial exploration missions. The five areas covered are: (i) field camp environment and activities, (ii) the engineering processes surrounding the hot water drilling, (iii) sample handling, including recovery, stability and preservation, (iv) clean access methodologies and removal of sample material, and (v) the biodiversity and distribution of bacteria around the Antarctic. Comparisons are made between the microbiology of the Lake Ellsworth field site and other Antarctic systems, including the lakes on Signy Island, and on the Antarctic Peninsula at Lake Hodgson. Ongoing research to better define and characterize the behaviour of natural and introduced microbial populations in response to deep-ice drilling is also discussed. We recommend that future access programmes: (i) assess each specific local environment in enhanced detail due to the potential for local contamination, (ii) consider the sterility of the access in more detail, specifically focusing on single cell colonization and the introduction of new species through contamination of pre-existing microbial communities, (iii) consider experimental bias in methodological approaches, (iv) undertake in situ biodiversity detection to mitigate risk of non-sample return and post-sample contamination, and (v) address the critical question of how important these microbes are in the functioning of Antarctic ecosystems.

Adaptation of an osmotically pumped continuous in situ water sampler for application in riverine environments.

We present the design of an osmotic water sampler that is adapted to and validated in freshwater. The sample is drawn into and stored in a continuous narrow bore tube. This geometry and slow pump rate (which is temperature dependent: 0.8 mL/d at 4 °C to 2.0 mL/d at 28 °C) minimizes sample dispersion. We have implemented in situ time-stamping which enables accurate study of pump rates and sample time defining procedures in field deployments and comparison with laboratory measurements. Temperature variations are common in rivers, and without an accurate time-stamping, or other defining procedure, time of sampling is ambiguous. The sampler was deployed for one month in a river, and its performance was evaluated by comparison with manually collected samples. Samples were analyzed for major ions using Ion Chromatography and collision reaction Inductively Couple Mass Spectrometry. Despite the differences of the two sampling methods (osmotic sampler averages, while manual samples provide snapshots), the two data sets show good agreement (average R(2) ≈ 0.7), indicating the reliability of the sampler and at the same time highlighting the advantages of high frequency sampling in dynamic environments.

Chemically resistant microfluidic valves from Viton® membranes bonded to COC and PMMA.

We present a reliable technique for irreversibly bonding chemically inert Viton® membranes to PMMA and COC substrates to produce microfluidic devices with integrated elastomeric structures. Viton® is widely used in commercially available valves and has several advantages when compared to other elastomeric membranes currently utilised in microfluidic valves (e.g. PDMS), such as high solvent resistance, low porosity and high temperature tolerance. The bond strength was sufficient to withstand a fluid pressure of 400 kPa (PMMA/Viton®) and 310 kPa (COC/Viton®) before leakage or burst failure, which is sufficient for most microfluidic applications. We demonstrate and characterise on-chip pneumatic Viton® microvalves on PMMA and COC substrates. We also provide a detailed method for bonding fluorinated Viton® elastomer, a highly chemically compatible material, to PMMA and COC polymers. This allows the production of microfluidic devices able to handle a wide range of chemically harsh fluids and broadens the scope of the microfluidic platform concept.

Temporal optimization of microfluidic colorimetric sensors by use of multiplexed stop-flow architecture.

We present two microfluidic architectures (continuous flow and multiplexed stop flow) for miniaturized colorimetric nutrient sensors. These systems are compared with respect to the temporal response (for optimization of sampling rate) and reduction of reagent consumption. The continuous-flow system is capable of a sampling rate of 60 samples·h(-1), limited by Taylor dispersion. The novel multiplexed stop-flow (MSF) microsystem architecture is not limited by dispersion. A demonstration MSF system consisting of two stop-flow channels is presented. This requires 12.6 s to load each sample into a measurement channel and when scaled would be capable of a throughput of 285 h(-1) (with full color development). The MSF architecture is manufactured in PMMA/Viton/PMMA [where PMMA = poly(methyl methacrylate)], utilizes on-chip valving, and is scalable, thereby permitting sampling at much faster rates (subsecond). Either system is capable of remote deployment and continuous measurement of nutrient concentrations. The MSF system is particularly suited for applications requiring high temporal or spatial resolution; such as from moving vehicles.

Electroporation and lysis of marine microalga Karenia brevis for RNA extraction and amplification.

We describe here a simple device for dielectrophoretic concentration of marine microalga Karenia brevis non-motile cells, followed by electric field-mediated lysis for RNA extraction. The lysate was purified using magnetic beads and pure RNA extracted. RNA quality was assessed off-chip by nucleic acid sequence-based amplification and the optimum conditions for lysis were determined. This procedure will form part of an integrated microfluidic system that is being developed with sub-systems for performing cell concentration and lysis, RNA extraction/purification and real-time quantitative RNA detection. The integrated system and its components could be used for a large range of applications including in situ harmful algal bloom detection, transcriptomics and point-of-care diagnostics.

Determination of dissolved oxygen in the cryosphere: a comprehensive laboratory and field evaluation of fiber optic sensors.

Recent advances in the Cryospheric Sciences have shown that icy environments are host to consortia of microbial communities, whose function and dynamics are often controlled by the concentrations of dissolved oxygen (DO) in solution. To date, only limited spot determinations of DO have been possible in these environments. They reveal the potential for rates of change that exceed realistic manual sampling rates, highlighting the need to explore methods for the continuous measurement of DO concentrations. We report the first comprehensive field and laboratory performance tests of fiber-optic sensors (PreSens, Regensburg, Germany) for measuring DO in icy ecosystems. A series of laboratory tests performed at low and standard temperatures (-5 to 20 °C) demonstrates high precision (0.3% at 50 µmol/kg and 1.3% at 300 µmol/kg), rapid response times (<20 s), and minimal drift (<0.4%). Survival of freeze thaw was problematic, unless the sensor film was mechanically fixed to the fiber and protected by a stainless steel sheath. Results of two field deployments of sensors to the Swiss Alps and Antarctica largely demonstrate a performance consistent with laboratory tests and superior to traditional methods.

Discrimination and analysis of phytoplankton using a microfluidic cytometer.

Identification and analysis of phytoplankton is important for understanding the environmental parameters that are influenced by the oceans, including pollution and climate change. Phytoplanktons are studied at the single cell level using conventional light-field and fluorescence microscopy, but the technique is labour intensive. Flow cytometry enables rapid and quantitative measurements of single cells and is now used as an analytical tool in phytoplankton analysis. However, it has a number of drawbacks, including high cost and portability. We describe the fabrication of a microfluidic (lab-on-a-chip) device for high-speed analysis of single phytoplankton. The device measures fluorescence (at three wavelength ranges) and the electrical impedance of single particles. The system was tested using a mixture of three algae (Isochrysis Galbana, Rhodosorus m., Synechococcus sp.) and the results compared with predictions from theory and measurements using a commercial flow cytometer (BD FACSAria). It is shown that the microfluidic flow cytometer is able to distinguish and characterise these different taxa and that impedance spectroscopy enables measurement of phytoplankton biophysical properties.